Genetic engineering of bacteria is well-known, however one such method, transduction, is less practiced. Transduction involves the use of a bacterial virus (or bacteriophage) to deliver DNA (contained in the virus) into the cell it infects. Inserting a desired sequence of base pairs in a phage is considerably easier than doing so in a bacteria since the DNA molecule of a bacteriophage is relatively small when compared with that of a bacteria. Therefore, if the DNA molecule of a bacteriophage is "engineered" by manipulation of its sequence, and the bacteriophage then intentionally introduced to a population of bacteria under conditions promoting infection and consequent transduction, the bacteria can be "genetically engineered" in a relatively simple process.
Conventional genetic engineering utilizes the transfer of plasmids, a less stable method of changing the DNA composition of an organism than transduction, which results in the desired DNA sequence being inserted directly into the host chromosome. Therefore, in the latter case one is assured that the inserted genetic material will be carried to subsequent generations after replication.
One of the environments in which bacteria flourish and potentially are of great economic benefit or detriment, is in acidic ambient temperature environments, such as mined ore or coal, tailings dumps, or the like. Such accumulations of ore, coal or tailings conventionally occur in large quantities, sometimes involving many thousands of tons spread over many acres.
Three related microorganism-based processes are all relevant to these environments and the present invention: first, the removal of sulfur from coal prior to its burning (thereby reducing subsequent release of sulfur oxides), secondly, the "biomining" of valuable metals from mined ore deposits, and thirdly, the prevention of acid leaching from tailings deposits of processed ore and from abandoned coal or metal mines. Each of these phenomena may be accomplished by introducing bacteriophage DNA into appropriate bacteria to effect the desired process. By placing genetically engineered organisms into the appropriate location in the first two processes noted above, or by placing selected bacteriophage into the acidic environment under conditions that promote infection and lysis of the resident bacteria in the third process, the response of the bacteria to their environment can be altered.
For instance, naturally occurring bacteria such as Thiobacillus ferrooxidans are found in sulfur-containing coal deposits. Thiobacillus readily attacks inorganic (pyritic) sulfur but not organic sulfur. Thus far, no microorganism has been found which is capable of removing both organic and inorganic forms of sulfur from coal--complete microbiological removal of sulfur from coal may be currently accomplished only by sequential treatment using completely different conditions. The time, expense and technical difficulties associated with such a two step procedure renders it impractical and uneconomic, and it is not currently practiced.
Likewise, acidic leaching from tailings piles and abandoned coal and metal mines at pHs of from 1.5 to bout 5.0 occurs because of sulfide oxidation by Thiobacillus sp ultimately resulting in sulfuric acid. Procedures are currently available to either kill such bacteria or render them inactive, but such procedures involve the application of chemicals (such as sodium dodecyl sulfate alone or combined with organic acids) in large quantities to the tailings piles, which are expensive and are in and of themselves environmentally objectionable. The introduction of a naturallyoccurring biological control agent, such as a bacteriophage, would be a preferable method of controlling acid leaching.
Finally, acidophilic heterotrophic bacteria which normally inhabit valuable metal-containing ores are tolerant of the normally toxic metals and low pHs associated with such environments, but do not have the genetic capability to enable these bacteria to "leach" the valuable metal. Such leaching may occur in a number of ways, but primarily through the microbial production of sulfuric acid in situ which leaches many metals from the surrounding ore. A bacteria capable of surviving such conditions which is genetically engineered to render such valuable metals more readily available for extraction would reduce the significant costs incurred in processing huge quantities of ore for recovery of relatively small amounts of the particular valuable metals.
For example, biooxidation of refractory arsenical sulfide concentrates for gold recovery indicates that recovery of available gold can be increased from about 66 percent using cyanide alone, to about 95 percent when used in conjunction with biological processes. The rate of biooxidation of ores is governed by substrate concentration, accessibility and the amount and specific activity of the biologically-produced enzymes involved. The amount of enzyme is usually proportional to the biomass, which is a result of the rate of growth of the microorganism. The processes as contemplated herein do not generate significant amounts of heat and thus would use naturally-occurring heterotrophic bacteria, rather than the thermophilic bacteria used in some industrial processes.
Heretofore, researchers in this area have failed to identify bacterial viruses capable of infecting and, thereby, potentially genetically engineering, bacteria found in such highly acidic environments. It is believed by some that such procedures are not possible at this time. Therefore, it is an object of this invention to provide, specifically, a bacteriophage capable of infecting acidophilic heterotrophic bacteria so as to deliver a desired DNA sequence into the host bacteria. It is a further object of the invention to provide processes whereby such genetically engineered bacteria are enabled to perform a function heretofore impossible.